Biological specimens or materials are often investigated microscopically. Appropriate objects can especially be investigated with wide field optics which image the object or a plane of the object onto a position-resolving detector. The investigation can, for example, take place with conventional microscopy or fluorescence microscopy, especially quantitative fluorescence microscopy. The objects can especially be biochips which were produced with photolithographic techniques or by means of a spotter or the objects can be material surfaces.
There is always the problem of the presence of stray light when using wide field optics for imaging heterogeneously luminescing surfaces or areal objects onto a position-resolving detector, for example, a CCD-camera. The stray light negatively affects the imaging of the object. Stray light is here understood to mean all light which reduces or makes incorrect the contrast of the detected intensity distribution. Stray light arises, for example, because of reflections and scattered light on surfaces, in glasses (for example, because of air pockets), because of inherent fluorescence of the used glasses, on frames or, in fluorescence measurements, because of non-suppressed excitation light. Furthermore, stray light can also come from regions of the object or the specimen lying outside of the focus plane, for example, from the backside of an object carrier. Stray light is especially a problem when the brightness distribution of the specimen is intensely heterogeneous and a high contrast ratio is required for the detection.
For example, in an image of an object, which includes a multiplicity of like fluorescing spheres, a bright surface can be formed from a corresponding plurality of like fluorescing spheres or circles having sharply defined diameters. Apart from diffraction effects, in the ideal case, maximum brightness should be present where the spheres or circles are disposed. In the remaining regions, complete darkness should be present. Actually, however, where it should be dark, a certain residual brightness is present, that is, a non-homogeneous background whose cause lies in the above-mentioned stray light. If this background were distributed homogeneously over the image, it could be viewed as an offset and could be subtracted from all pixel values of the detector in a simple manner. Such an ideal case is, however, hardly to be found in practice and accordingly, this procedure cannot generally be applied.
In radiometric measurements, specifically in biochip applications and in quantitative fluorescence microscopy in general, the useable intensity dynamic, which reflects the ratio of the largest to the smallest detectable value, is greatly limited by the non-homogeneous background because, for an unknown fluorophor distribution, the useful light cannot be distinguished from the stray light. This is especially problematical when no or hardly any reference areas are available on which locally the background can be determined, that is, for example, in high density biochips which are produced photolithographically.
A possibility for avoiding stray light lies in the use of confocal laser scanners. In confocal laser scanners, always only a small area of the specimen of a few μm2 is illuminated and only this small surface is viewed with the detection. Stray light is suppressed ab initio if this is carried out consequently with the aid of a well-adapted pinhole diaphragm. Laser scanners have, however, several disadvantages compared to microscopes with wide field optic. For example, in fluorescence microscopy, excitation saturation and a strong blanching of fluorophores can occur because of the high radiation intensity in the focus. Further, there are significant restrictions in the selection of the wavelength. Additional disadvantages are: many movable components, a high adjustment complexity and a low quantum efficiency of the detector which, as a rule, is a photomultiplier.
A method and an arrangement for the depth selection of microscope images is described in German patent publication 199 30 816. In this method and arrangement, a one-dimensional periodic grating (for example, a striped grating) is used for illumination. At least n (n>2) CCD-camera recordings are made. The structure of the illumination is shifted in each case by 1/n of the grating constant. From the at least three recordings, a confocal section of the specimen is thereafter computed. This method is subject to artefacts when the grating does not generate a sinusoidally-shaped illumination intensity on the specimen.
U.S. Pat. No. 6,376,818 discloses an imaging system and imaging method for microscopes wherein a structured illumination by means of superposition of two coherent light beams is provided. The method, like the method of German patent publication 199 30 816, has primarily the objective of generating optical sections in different object planes in the same manner as in a laser scanning microscope.
Both methods pursue the objective to obtain a depth resolution of thick specimens. The methods function to obtain confocal sections of a specimen or of an object, which is thick in comparison to the depth of field, with a wide field optic. In both cases, the computation complexity is relatively,large because trigonometric equations must be solved.
Published German patent application 103 30 716.8 discloses an arrangement for carrying out a method of eliminating stray light in the imaging of heterogeneous, luminous or illuminated flat objects. The arrangement includes a beam source having a downstream illumination optic homogenizing the beam for homogeneously illuminating a downstream field diaphragm plane wherein a structured field diaphragm is mounted for generating an illumination structure superposed on the object or specimen. This illumination structure is imaged via first optic means onto the specimen. This first optic means can include an illumination tube, a color divider as may be required and an objective. In addition, second optical means are provided for imaging the specimen together with the superposed illumination structure onto a position-resolving detector, especially, for optical radiation. The arrangement further includes adjusting means with which the illumination structure can be positioned in a defined manner in the object plane on the object or on the specimen. The detector is connected to an evaluation unit for determining and eliminating the stray light. A structured bright field illumination with at least two different illumination patterns is used wherein dark regions do not overlap. From corresponding images, a dark image and a bright image can be determined. The resulting image can be obtained by subtracting the dark image from the bright image.
The structured bright field illumination is provided by this arrangement. With this bright field illumination, the object illumination and the imaging of the object take place together with the illumination of the field diaphragm structure via a single objective. The bright field illumination can cause the excitation light in the objective to bring about the occurrence of stray light, especially, because of inherent fluorescence of the glasses used. Furthermore, the rear side of an object (for example, a biochip) is radiated with almost the same excitation intensity as the focus plane. For this reason, the fluorescence intensity, which is caused by the contamination on the rear side, can also be correspondingly high and lead to measurement errors. It was therefore suggested in a second method for avoiding these disadvantages that a structured dark field illumination be used in lieu of the bright field illumination.
With these two methods, it is, however, necessary to carry out an interpolation between non-illuminated regions in order to obtain a complete dark light image, that is, stray light image.